The term age-related macular degeneration (AMD) is used to describe a particular phenotype that commonly occurs in patients over the age of 60 and is the leading cause of blindness in the elderly in developed countries. The mechanism by which cell damage occurs in AMD is unknown. It is likely that AMD is a polygenic disease and there may be multiple unrelated mechanisms involved. However, it is also possible that multiple gene defects lead to a final common pathway through which cell damage occurs. We hypothesize that gene expression changes associated with oxidative damage play a critical role in photoreceptor degeneration. Exposure of mice to high levels of inspired oxygen causes photoreceptor cell damage almost certainly from oxidative damage. We propose to study the gene expression changes that occur in the retinas of mice exposed to high levels of oxygen and compare them to changes that occur in other purported models of AMD. Abcr null mice provide a model of lipofuscin accumulation in RPE cells, and oxidative stress that may be relevant to AMD. Prolonged light exposure is purported to cause AMD phenotype through oxidative damage. Expression of fibroblast growth factor 2 (FGF2) or brain-derived neurotrophic factor (BDNF) in the retina prevents hyperoxia-induced retinal degeneration. We plan to determine the gene expression changes in photoreceptors and RPE of mice in the above models. The innovative nature of our proposal involves using microarray analysis to screen for multiple genes that may be involved in the process of oxidative damage and photoreceptor degeneration. Laser capture microdissection is performed for single cell isolation of photoreceptors and RPE cells. RNA is isolated and hybridized with a custom-made 5,376 clone set cDNA library, to look for patterns of gene expression. The laboratory has already created this clone set, representing available mouse genes known or postulated to be involved in degeneration and cell stress responses, and successfully tested the feasibility of our microarray approach. Single gene expression is then confirmed using real-time PCR. Understanding the pattern of gene expression changes may provide mechanistic information regarding the molecular signals leading from oxidative stress to cell death in models of AMD and thereby provide new targets for therapeutic intervention. Research training will focus on gaining a detailed understanding of the pathogenesis of AMD under the mentorship of Dr. Peter A. Campochiaro and Dr. Donald J. Zack at the Wilmer Eye Institute of the Johns Hopkins University. The major emphasis of the career development will be basic science bench research as well as a program of mentorship, didactic lectures and coursework, and group studies and conferences. The goal is to obtain solid scientific training that would enable me to establish, oversee, and sustain a significant R01 level program of research.
